Cystic Fibrosis (CF) is caused by deleterious mutations in the gene that codes for the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-activated anion channel. Numerous organ systems are affected in CF, but death typically results from the lung disease which is typified by the production of unusually thick mucus, severe chronic bacterial infections, and bronchiectasis. Unfortunately, the underlying sequence of events which leads to this pattern of pathology is unclear and controversial. Our longstanding hypothesis is that CFTR mediates liquid secretion by tracheobronchial submucosal glands which is important for hydrating the macromolecular component of gland secretion and flushing this material through the gland ducts to the airway surface. Thereby, in CF, loss of CFTR function would lead to secretion of overly thick mucus from glands and depletion of periciliary fluid at the airway surface. Disruption of mucociliary transport thus occurs, predisposing the airways to bacterial colonization. Our previous studies of pig airways demonstrated that inhibition of anion and liquid secretion reproduces much of the lung pathology of CF including mucus occlusion of submucosal glands, secretion of inspissated mucus, depletion of periciliary fluid, and impaired mucociliary transport. With the present application, we propose a series of studies which we anticipate will strengthen this hypothesis. Using pig airways as a model, we will define critical aspects of the intracellular signal transduction elements for physiologically important endogenous secretogogues that will help define how secretion is regulated, clarify the role played by CFTR, and identify possible targets for therapeutic manipulation. We will obtain and study human CF airways to document which gland secretion pathways are disabled or intact in this disease. We will pursue development of an in vitro technique to model CF in porcine airways by utilizing shRNA to reduce CFTR expression in explants of airway tissue. Using anion and liquid secretion inhibitors to model the CF defect in pigs airways, we will examine the consequences of thick mucus on Pseudomonas aeruginosa colonization of the airway mucosa. We believe that these studies will provide crucial new information regarding the role of airway glands in the pulmonary pathogenesis of CF airway disease. Without doubt, development of future treatment strategies for CF will depend upon a better understanding of the underlying causes of the disease process.